PROBLEM: Septic lung injury is an often-fatal complication of numerous clinical conditions, and affects a significant percentage of the VA patient population. This injury is characterized by gross cellular damage to the lung alveoli, which allows red cells and plasma to enter the alveolar air space. This acute injury often leads to permanent scarring of the lung. It is widely believed that cytotoxic agents released by neutrophils, the white blood cells that invade the lung during sepsis, cause the initial alveolar injury. However, the trigger that causes this neutrophil invasion is unknown. We hypothesize that the trigger is microthrombus formation within alveolar capillaries early in sepsis, and further hypothesize that agents that prevent microthrombus formation may prevent the neutrophil invasion and minimize the injury. SPECIFIC AIMS: 1) Measure thrombus formation within lung microvessels early in sepsis, and measure their effects on the distribution of alveolar perfusion: We will use a clinically relevant live bacteria model to induce sepsis in rats, and use immunofluorescence to measure microthrombus formation in alveolar capillaries. At various times during sepsis development, we will correlate microthrombus location with trapping patterns of small-diameter fluorescent latex particles infused into lung capillaries to determine the association between microthrombus formation and alveolar perfusion, which we have shown is markedly mal-distributed in sepsis. 2) Show that neutrophil sequestration into the lung occurs only after microthrombus formation: We will use immunofluorescence to label microthrombi and neutrophils in lung tissue at various times after sepsis induction, to show that microthrombus formation precedes neutrophil invasion. 3) Show that prevention of thrombus formation prevents neutrophil sequestration, and prevents lung injury: low molecular weight heparin, recombinant hirudin, and recombinant activated protein C have anti-thrombotic properties that prevent microthrombus formation. We therefore aim to show if administration of these agents in sepsis prevents microthrombus formation and prevents subsequent neutrophil invasion of the lungs. 4) Determine the role of thromboxane in control of capillary perfusion during sepsis: thromboxane alters lung microvascular perfusion in a manner similar to sepsis, and is known to be secreted by neutrophils. We will use immunofluorescence to label thromboxane and neutrophils in the lungs of septic rats, then measure perfusion distribution in neutrophil- and thromboxane-positive regions to determine if regions positive for neutrophils and thromboxane have less perfusion than regions with fewer neutrophils. POTENTIAL IMPACT ON VETERANS'HEALTH CARE: Sepsis can occur as a result of abdominal combat injuries, a perforated bowel, inflammatory bowel disease, or other surgical complications. The resulting lung injury often causes these patients to experience very high rates of mortality. Our proposed studies therefore address a highly veteran-centric clinical problem, and have great potential to improve veterans'health care. PUBLIC HEALTH RELEVANCE: Narrative (Relevance Statement) Identifying the initial event that initiates lung injury in sepsis may be the best way to address methods to prevent this devastating and often fatal disease. Unfortunately, this is a subject about which little is known. Our studies will provide new insight into this fundamental aspect of the lung's reaction to sepsis. We believe this is crucial to the development of successful therapeutic strategies for VA patients. VA data show that approximately 10,000 patients are stricken annually with ARDS, respiratory failure, and pulmonary edema throughout the VA system. These are conditions that are frequently caused by sepsis-associated lung injury. These often-fatal conditions effect lung endothelial and epithelial barrier function in ways that allow plasma and whole blood to enter the alveolar air space. If we can identify the initial incident that begins the cascade of events that characterize lung injury in sepsis, we have a better chance of developing effective treatments for these often-fatal conditions. This is the goal of our proposed studies.